Novel surface exposed proteins from chlamydia pneumoniae

ABSTRACT

The invention relates to the identification of members of a gene family from the human respiratory pathogen  Chlamydia pneumoniae , encoding surface exposed membrane proteins of a size of approximately 89-101 kDa, preferably about 89.6-100.3 kDa and about 56.1 kDa. The invention relates to the novel DNA sequences, the deduced amino acid sequences of the corresponding proteins and the use of the DNA sequences and the proteins in diagnosis of infections caused by  C. pneumoniae , in pathology, in epidemiology, and as vaccine components.

The present invention relates to the identification of members of a genefamily from the human respiratory pathogen Chlamydia pneumoniae,encoding surface exposed membrane proteins of a size of approximately89-101 kDa and of 56-57 kDa, preferably about 89.6-100.3 kDa and about56.1 kDa. The invention relates to the novel DNA sequences, the deducedamino acid sequences of the corresponding proteins and the use of theDNA sequences and the proteins in diagnosis of infections caused by C.pneumoniae, in pathology, in epidemiology, and as vaccine components.

GENERAL BACKGROUND

C. pneumoniae is an obligate intracellular bacteria (Christiansen andBirkelund (1992); Grayston et al. (1986)). It has a cell wall structureas Gram negative bacteria with an outer membrane, a periplasmic space,and a cytoplasmic membrane. It is possible to purify the outer membranefrom Gram negative bacteria with the detergent sarkosyl. This fractionis named the ‘outer membrane complex (OMC)’ (Caldwell et al. (1981)).The COMC (Chlamydia outer membrane complex) of C. pneumoniae containsfour groups of proteins: A high molecular weight protein 98 kDa asdetermined by SDS-PAGE, a double band of the cysteine rich outermembrane protein 2 (Omp2) protein of 62/60 kDa, the major outer membraneprotein (MOMP) of 38 kDa, and the low-molecular weight lipo-protein Omp3of 12 kDa. The Omp2/Omp3 and MOMP proteins are present in COMC from allChlamydia species, and these genes have been cloned from both C.trachomatis, C. psittaci and C. pneumoniae. However, the gene encoding98 kDa protein from C. pneumoniae COMC have not been characterized orcloned.

The Current State of C. pneumoniae Serology and Detection

C. pneumoniae is an obligate intra-cellular bacteria belonging to thegenus Chlamydia which can be divided into four species: C. trachomatis,C. pneumoniae, C. psittaci and C. pecorum. Common for the four speciesis their obligate intra cellular growth, and that they have a biphasiclife cycle, with an extracellular infectious particle (the elementarybody, EB), and an intercellular replicating form (the reticulate body,RB). In addition the Chlamydia species are characterized by a commonlipopolysaccharide (LPS) epitope that is highly immunogenic in humaninfection. C. trachomatis is causing the human ocular infection(trachoma) and genital infections. C. psittaci is a variable group ofanimal pathogens where the avian strains can occasionally infect humansand give rise to a severe pneumonia (ornithosis). The first C.pneumoniae isolate was obtained from an eye infection, but it wasclassified as a non-typable Chlamydia. Under an epidemic outbreak ofpneumonia in Finland it was realized that the patients had a positivereaction in the Chlamydia genus specific test, (the lygranum test), andthe patients showed a titre increase to the untyped Chlamydia isolates.Similar isolates were obtained in an outbreak of upper respiratory tractinfections in Seattle, and the Chlamydia isolates were classified as anew species, Chlamydia pneumoniae (Grayston et al. (1989)). In addition,C. pneumoniae is suggested to be involved in the development ofatherosclerotic lesions and for initiating bronchial asthma (Kuo et al.(1995)). These two conditions are thought to be caused by either chronicinfections, by a hypersensitivity reaction, or both.

Diagnosis of Chlamydia pneumoniae Infections

Diagnosis of acute respiratory tract infection with C. pneumoniae isdifficult. Cultivation of C. pneumoniae from patient samples isinsensitive, even when proper tissue culture cells are selected for theisolation. A C. pneumoniae specific polymerase chain reaction (PCR) hasbeen developed by Campbell et al. (1992).

Even though Chlamydia pneumoniae has in several studies been detected bythis PCR it is debated whether this method is suitable for detectionunder all clinical situations. The reason for this is, that the cellscarrying Chlamydia pneumoniae in acute respiratory infections have notbeen determined, and that a chronic carrier state is expected but it isunknown in which organs and cells they are present. Furthermore, the PCRtest is difficult to perform due to the low yield of these bacteria anddue to the presence of inhibitory substances in the patient samples.Therefore, it will be of great value to develop sensitive and specificsero-diagnostics for detecting both acute and chronic infections.Sero-diagnosis of Chlamydia infections is currently based on eithergenus specific tests as the Lygranum test and ELISA, measuring theantibodies to LPS, or the more species specific tests where antibodiesto purified EBs are measured by microimmuno fluorescence (Micro-IF)(Wang et al. (1970)). However, the micro-IF method is read bymicroscopy, and in order to ensure correct readings the result must becompared to the results with C. trachomatis used as antigen due to thecross-reacting antibodies to the common LPS epitope. Thus, there existsin the art an urgent need for development of reliable methods forspecies specific diagnosis of Chlamydia pneumoniae, as has beenexpressed in Kuo et al. (1995); “ . . . a rapid reliable laboratory testof infection for the clinical laboratory is a major need in the field”.Furthermore, the possible involvement of C. pneumoniae inatherosclerosis and bronchial asthma clearly warrants the development ofan effective vaccine.

DETAILED DISCLOSURE OF THE INVENTION

The present invention aims at providing means for efficient diagnosis ofinfections with Chlamydia pneumoniae as well as the development ofeffective vaccines against infection with this microorganism. Theinvention thus relates to species specific diagnostic tests forinfection in a mammal, such as a human, with Chlamydia pneumoniae, saidtests being based on the detection of antibodies against surface exposedmembrane proteins of a size of approximately 89-101 kDa and of 56-57kDa, preferably of about 89.6-100.3 kDa and about 56.1 kDa (the range insize of the deduced amino acid sequences was from 100.3 to 89.6 exceptfor Omp13, with the size of 56.1 kDa), or the detection of nucleic acidfragments encoding such proteins or variants or subsequences thereof.The invention further relates to the amino acid sequences of proteinsaccording to the invention, to variants and subsequences thereof, and tonucleic acid fragments encoding these proteins or variants orsubsequences thereof. The present invention further relates toantibodies against proteins according to the invention. The inventionalso relates to the use of nucleic acid fragments and proteins accordingto the invention in diagnosis of Chlamydia pneumoniae and vaccinesagainst Chlamydia pneumoniae.

Prior to the disclosure of the present invention only a very limitednumber of genes from C. pneumoniae had been sequenced. These wereprimarily the genes encoding known C. trachomatis homologues: MOMP,Omp2, Omp3, Kdo-transferase, the heat shock protein genes GroEl/Es andDnaK, a ribonuclease P homologue and a gene encoding a 76 kDa protein ofunknown function. The reason why so few genes have been cloned to dateis the very low yield of C. pneumoniae which can be obtained afterpurification from the host cells. After such purification the DNA mustbe purified from the EBs, and at this step the C. pneumoniae DNA caneasily be contaminated with host cell DNA. In addition to these inherentdifficulties, it is exceedingly difficult to cultivate C. pneumoniae anduse DNA technology to produce expression libraries with very low amounts(few μg of DNA. It has been known since 1993 (Melgosa et al., 1993, thata 98 kDa protein is present in OMC from C. pneumoniae. Even though theprotein bands of 98 kDa was mentioned to be part of the OMC of C.pneumoniae by Melgosa, the gene sequences and thus the deduced aminoacid sequences have not been determined. Only bands originating fromChlamydia pneumoniae proteins in general separated by SDS-PAGE aredescribe therein.

However, the gene encoding this protein has not been determined beforethe present invention. Only a very weak or no reaction with patient seracan be observed to the 98 kDa protein (Campbell et al. 1990) and priorto the work of the present inventors it has not been recognized that the89-101 kDa proteins are surface exposed or that they in fact isimmunogenic (see below). In this report it is described that a number ofhuman serum samples reacts with a C. pneumoniae protein that in SDS-PAGEmigrate as 98 kDa. The protein was not further characterized and it istherefore not in conflict with the present application.

Campbell et al. (1990) described that sera from four patients from whichChlamydia pneumonia was isolated reacted with bands of 98 kDa inimmunoblotting using whole-cell lysates. They also showed that noproteins with similar molecular weights were recognised by serum samplesin either Chlamydia trachomatis or Chlamydia psittaci and they thereforesuggest that the protein present in the 98 kDa band could be used as apotential diagnostic tool for the recognition of Chlamydia pneumoniaeinfection. The protein content within the 98 kDa region was not furthercharacterised and its localisation within the Chlamydia was not shown.

Halme et al. (1997) described the presence of human T-cell epitopes inC. pneumoniae proteins of 92-98 kDa. The proteins were eluted fromSDS-PAGE of total chlamydia proteins but the identity of the proteinswere not determined.

Use of antibodies to screen expression libraries is a well known methodto clone fragments of genes encoding antigenic parts of proteins.However, since patient sera do not show a significant reaction with the98 kDa protein it has not been possible to use patient serum to clonethe proteins.

It was known that monoclonal antibodies generated by the inventorsreacted with conformational epitopes on the surface of C. pneumoniae andthat they also reacted with C. pneumoniae OMC by immuno-electronmicroscopy (Christiansen et al. 1994). Furthermore, the 98 kDa proteinis the only unknown protein from the C. pneumoniae OMC (Melgosa et al.1993). The present inventors chose to take an unconventional step inorder to clone the gene encoding the hitherto unknown 98 kDa protein: C.pneumoniae OMC was purified and the highly immunogenic conformationalepitopes were destroyed by SDS-treatment of the antigen beforeimmunization. Thereby an antibody (PAB 150) to less immunogenic linearepitopes was obtained. This provided the possibility to obtain anantiserum which could detect the protein, and it was shown that a genefamily encoding the 89-101 kDa and 56 proteins according to theinvention could be detected in colony blotting of recombinant E. coli.

Mice infected with C. pneumoniae generate antibodies to the proteinsidentified by the inventors and named Omp4-15, but do not recognize theSDS treated heat denatured antigens normally used for SDS-PAGE andimmunoblotting. However, a strong reaction was seen if the antigen wasnot heat denatured. It is therefore highly likely that if a similarreaction is seen in connection with human infections the antigens of thepresent invention will be of invaluable use in sero-diagnostic tests andmay very likely be used as a vaccine for the prevention of infections.

By generating antibodies against COMC from C. pneumoniae a polyclonalantibody (PAB 150) was obtained which reacted with all the proteins.This antibody was used to identify the genes encoding the 89.6-101.3 kDaand 56.1 kDa proteins in an expression library of C. pneumoniae DNA. Aproblem in connection with the present invention was that a familycomprising a number of similar genes were found in C. pneumoniae.Therefore, a large number of different clones were required to identifyclusters of fragments. Only because the rabbit antibody generated by theuse of SDS-denatured antigens contained antibodies to a high number ofdifferent epitopes positioned on different members of the protein familydid the inventors succeed in cloning and sequencing four of the genes.One gene was fully sequenced, a second was sequenced except for thedistal part and shorter fragments of two additional genes were obtainedby this procedure. To obtain the DNA-sequence of the additional genesand to sear for more members of the gene family long range PCR withprimers derived from the sequenced genes, and primers from the genesalready published in the database were used. This approach gave rise tothe detection of additional eight genes belonging to this family. Thegenes were situated in two gene clusters: Omp12, 11, 10, 5, 4, 13 and 14in one cluster and Omp6, 7, 8, 9 and 15 in the second. Full sequence wasobtained from Omp4, 5, 6, 7, 8, 9, 10, 11 and 13, and partial sequenceof Omp12, 14. Omp13 was a truncated gene of 1545 nucleotides. The restof the full length genes were from 2526 (Omp7) to 2838 (Omp15)nucleotides. The deduced amino acid sequences revealed putativepolypeptides of 89.6 to 100.3 kDa, except for Omp13 of 56.1 kDa.Alignment of the deduced amino acid sequences showed a maximum identityof 499 (Omp5/Omp9) when all the sequences were compared. Except forOmp13, the lowest homology was to Omp7 with no more than 34% identity toany of the other amino acid sequences. The scores for Omp13 was from29-32% to all the other sequences.

In the present context SEQ ID Nos. 1 and 2 correspond to Omp4, SEQ IDNos 3 and 4 correspond to Omp5, SEQ ID Nos 5 and 6 correspond to Omp6,SEQ ID Nos 7 and 8 correspond to Omp7, SEQ ID Nos 9 and 10 correspond toOmp8, SEQ ID Nos 11 and 12 correspond to Omp9, SEQ ID Nos 13 and 14corresponds to Omp10, SEQ ID Nos 15 and 16 corresponds to Omp11, SEQ IDNos 17 and 18 corresponds to Omp12, SEQ ID Nos 19 and 20 corresponds toOmp13, SEQ ID Nos 21 and 22 corresponds to Omp14, and SEQ ID Nos 23 and24 corresponds to Omp15.

The estimated size of the Omp proteins of the of the present inventionare listed in the following. Omp 4 has a size of 98.9 kDa, Omp5 has anestimated size of 97.2 kDa, Omp6 has an estimated size of 100.3 kDa,Omp7 has an estimated size of 89.7 kDa, Omp8 has an estimated size of90.0 kDa, Omp9 has an estimated size of 96.7 kDa, Omp10 has an estimatedsize of 98.4 kDa, Omp11 has an estimated size of 97.6 kDa, Omp13 has anestimated size of 56.1 kDa, Omp 12 and 14 being partial.

Furthermore, SEQ ID No 25 is a subsequence of SEQ ID No 3, SEQ ID No 26is a subsequence of SEQ ID No 4, SEQ ID No 27 is a subsequence of SEQ IDNo 5, SEQ ID No 28 is a subsequence of SEQ ID No 6, SEQ ID No 29 is asubsequence of SEQ ID No 7, and SEQ ID No 30 is a subsequence of SEQ IDNo 8.

Part of the omp proteins were expressed as fusion proteins, and micepolyclonal monospecific antibodies against the proteins were produced.The antibodies reacted with the surface of C. pneumoniae in bothimmunofluorescence and immunoelectron microscopy. This shows for thefirst time that the 89-101 kDa and 56-57 kDa protein family in C.pneumoniae comprises surface exposed outer membrane proteins. Thisimportant finding leads to the realization that members of the 89-101kDa and 56-57 kDa C. pneumoniae protein family are good candidates forthe development of a sero diagnostic test for C. pneumoniae, as well asthe development of a vaccine against infections with C. pneumoniae basedon using these proteins. Furthermore, the proteins may be used asepidemiological markers, and polyclonal monospecific sera against theproteins can be used to detect C. pneumoniae in human tissue or detectC. pneumoniae isolates in tissue culture. Also, the genes encoding the89-101 kDa and 56-57 kDa such as the 89.6-100.3 kDa and 56.1 proteinfamily may be used for the development of a species specific diagnostictest based on nucleic acid detection/amplification.

The full length Omp4 was cloned into an expression vector system thatallowed expression of the Omp4 polypeptide. This polypeptide was used asantigen for immunization of a rabbit. Since the protein was purifiedunder denaturing condition the antibody did not react with the nativesurface of C. pneumoniae, but it reacted with a 98 kDa protein inimmunoblotting where purified C. pneumoniae EB was used as antigen.Furthermore, the antibody reacted in paraffin embedded sections of lungtissue from experimentally infected mice.

A broad aspect of the present invention relates to a species specificdiagnostic test for infection of a mammal, such as a human, withChlamydia pneumoniae, said test comprising detecting in a patient orpreferable in a patient sample the presence of antibodies againstproteins from the outer membrane of Chlamydia pneumoniae, said proteinsbeing of a molecular weight of 89-101 kDa or 56-57 kDa, or detecting thepresence of nucleic acid fragments encoding said outer membrane proteinsor fragments thereof.

In the context of the present application, the term “patient sample”should be taken to mean an amount of serum from a patient, such as ahuman patient, or an amount of plasma from said patient, or an amount ofmucosa from said patient, or an amount of tissue from said patient, oran amount of expectorate, forced sputum or a bronchial aspirate, anamount of urine from said patient, or an amount of cerebrospinal fluidfrom said patient, or an amount of atherosclerotic lesion from saidpatient, or an amount of mucosal swaps from said patient, or an amountof cells from a tissue culture originating from said patient, or anamount of material which in any way originates from said patient. The invivo test in a human according to the present invention includes a skintest known in the art such as an intradermal test, e.g. similar to aMantaux test. In certain patients being very sensitive to the test, suchas is often the case with children, he test could be non-invasive, suchas a superficial test on the skin, e.g. by use of a plaster

In the present context, the term 89-101 kDa protein means proteinsnormally present in the outer membrane of Chlamydia pneumoniae, which inSDS-PAGE can be observed as one or more bands with an apparent molecularweight substantially in the range of 89-101 kDa. From the deduced aminoacid sequences the molecular size varies from 89.6 to 100.3 kDa.

Within the scope of the present invention are species specificsero-diagnostic tests based on the usage of the genes belonging to thegene family disclosed in the present application.

Preferred embodiments of the present invention relate to speciesspecific diagnostic tests according to the invention, wherein the outermembrane proteins have sequences selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, and SEQ ID NO: 24.

When used in connection with proteins according to the present inventionthe term “variant” should be understood as a sequence of amino acidswhich shows a sequence similarity of less than 100% to one of theproteins of the invention. A variant sequence can be of the same size orit can be of a different size as the sequence it is compared to. Avariant will typically show a sequence similarity of preferably at least50%, preferably at least 60%, more preferably at least 70%, such as atleast 80%, e.g. at least 90%, 95% or 98%.

The term “sequence similarity” in connection with sequences of proteinsof the invention means the percentage of identical and conservativelychanged amino acid residues (with respect to both position and type) inthe proteins of the invention and an aligned protein of equal ofdifferent length. The term “sequence identity” in connection withsequences of proteins of the invention means the percentage of identicalamino acid with respect to both position and type in the proteins of theinvention and an aligned protein of equal of different length.

Within the scope of the present invention are subsequences of one of theproteins of the invention, meaning a consecutive stretch of amino acidresidues taken from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24. A subsequencewill typically comprise at least 100 amino acids, preferably at least 80amino acids, more preferably at least 70 amino acids, such as 50 aminoacids. It might even be as small as 10-50 amino acids, such as 20-40amino acids, e.g. about 30 amino acids. A subsequence will typicallyshow a sequence homology of at least 50%, preferably at least 60%, morepreferably at least 70%, such as at least 80%, e.g. at least 900, 95% or98%.

Diagnostic tests according to the invention include immunoassaysselected from the group consisting of a direct or indirect EIA such asan ELISA, an immunoblot technique such as a Western blot, a radio immunoassay, and any other non-enzyme linked antibody binding assay orprocedure such as a fluorescence, agglutination or precipitationreaction, and nephelometry.

A preferred embodiment of the present invention relates to speciesspecific diagnostic tests according to the invention, said testcomprising an ELISA, wherein antibodies against the proteins of theinvention or fragments thereof are detected in samples.

A preferred embodiment of the invention, is an ELISA based on detectionin samples of antibodies against proteins of the invention. The ELISAmay use proteins of the invention, or variants thereof, i.e. theantigen, as coating agent. An ELISA will typically be developedaccording to standard methods well known in the art, such as methodsdescribed in “Antibodies; a laboratory manual”, Ed. David Lane Harlow,Cold Spring Habor laboratories (1988), which is hereby incorporated byreference.

Recombinant proteins will be produced using DNA sequences obtainedessentially using methods described in the examples below. Such DNAsequences, comprising the entire coding region of each gene in the genefamily of the invention, will be cloned into an expression vector fromwhich the deduced protein sequence can be purified. The purifiedproteins will be analyzed for reactivity in ELISA using both monoclonaland polyclonal antibodies as well as sera from experimentally infectedmice and human patient sera.

From the experimentally infected mice sera it is known that non-linearepitopes are recognized predominantly. Thus, it is contemplated thatdifferent forms of purification schemes known in the art will be used toanalyze for the presence of discontinuous epitopes, and to analyzewhether the human immune response is also directed against suchepitopes.

Preferred embodiments of the present invention relate to speciesspecific diagnostic tests according to the invention, wherein thenucleic acid fragments have sequences selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID No:19, SEQ ID NO: 21, and SEQ ID NO.: 23.

In connection with nucleic acid fragments according to the presentinvention the term “variant” should be understood as a sequence ofnucleic acids which shows a sequence homology of less than 100%. Avariant sequence can be of the same size or it can be of a differentsize as the sequence it is compared to. A variant will typically show asequence homology of at least 50%, preferably at least 60%, morepreferably at least 70%, such as at least 80%, e.g. at least 90%, 95% or98%.

The term “sequence homology” in connection with nucleic acid fragmentsof the invention means the percentage of matching nucleic acids (withrespect to both position and type) in the nucleic acid fragments of theinvention and an aligned nucleic acid fragment of equal or differentlength.

In order to obtain information concerning the general distribution ofeach of the genes according to the present invention, PCR will beperformed for each gene on all available C. pneumoniae isolates. Thiswill provide information on the general variability of the genes ornucleic acid fragments of the invention. Variable regions will besequenced. From patient samples PCR will be used to amplify variableparts of the genes for epidemiology. Non-variable parts will be used foramplification by PCR and analyzed for possible use as a diagnostic test.It is contemplated that if variability is discovered, PCR of variableregions can be used for epidemiology. PCR of non-variable regions can beused as a species specific diagnostic test. Using genes encodingproteins known to be invariable in all known isolates prepared astargets for PCR to genes encoding proteins with unknown function.

Particularly preferred embodiments of the present invention, relate todiagnostic tests according to the invention, wherein detection ofnucleic acid fragments is obtained by using nucleic acid amplification,preferably polymerase chain reaction (PCR).

Within the scope of the present invention is a PCR based test directedat detecting nucleic acid fragments of the invention or variantsthereof. A PCR test will typically be developed according to methodswell known in the art and will typically comprise a PCR test capable ofdetecting and differentiating between nucleic acid fragments of theinvention. Preferred are quantitative competitive PCR tests or nestedPCR tests. The PCR test according to the invention will typically bedeveloped according to methods described in detail in EP B 540 588, EP A586 112, EP A 643 140 OR EP A 669 401, which are hereby incorporated byreference.

Within the scope of the present invention are variants and subsequencesof one of the nucleic acid fragments of the invention, meaning aconsecutive stretch of nucleic acids taken from SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23. Avariant or subsequence will preferably comprise at least 100 nucleicacids, preferably at least 80 nucleic acids, more preferably at least 70nucleic acids, such as at least 50 nucleic acids. It might even be assmall as 10-50 nucleic acids, such as 20-40 nucleic acids, e.g. about 30nucleic acids. A subsequence will typically show a sequence homology ofat least 30%, preferably at least 60%, more preferably at least 70%,such as at least 80%, e.g. at least 90%, 95% or 98%. The shorter thesubsequence, the higher the required homology. Accordingly, asubsequence of 100 nucleic acids or lower must show a homology of atleast 80%.

A very important aspect of the present invention relates to proteins ofthe invention derived from Chlamydia pneumoniae having amino acidsequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQID NO: 24 having a sequence similarity of at least 50%, preferably atleast 60%, more preferably at least 70%, such as at least 80%, e.g. atleast 90%, 95% or 98% and a similar biological function.

By the term “similar biological function” is meant that the proteinshows characteristics similar with the proteins derivable from themembrane proteins of Chlamydia pneumoniae. Such proteins compriserepeated motifs of GGAI (at least 2, preferable at least 3 repeats)and/or conserved positions of tryptophan, (w).

Comparison of the DNA sequences from genes encoding Omp4-15 shows thatthe overall similarity between the individual genes ranges between43-55%. Comparison of the amino acid sequences of Omp4-15 shows 34-49%identity and 53-64% similarity. The homology is generally scatteredalong the entire length of the deduced amino acids. However, as seenfrom FIG. 8 A-J there are some regions in which the homology is morepronounced. This is seen in the repeated sequence where the sequenceGGAI is repeated 4-7 times in the genes. It is interesting that the DNAhomology is not conserved for the sequences encoding the four aminoacids GGAI. This may indicate a functional role of this part of theprotein and indicates that the repeated structure did not occur by aduplication of the gene. In addition to the four amino acid repeats GGAIa region from amino acid 400 to 490 has a higher degree of homology thanthe rest of the protein, with the conserved sequence FYDPI occurring inall sequences. As further indication of similarity in function the aminoacid tryptophan (W) is perfectly conserved at 4-6 localizations in theC-terminal part of the protein.

Since none of the genes and deduced amino acid sequences of theinvention are identical the following is within the scope of the presentinvention; production of monospecific antibodies, the use of saidantibodies for characterizing which C. pneumoniae proteins areexpressed, the use of said antibodies for characterizing at which timeduring developmental life cycle said C. pneumoniae proteins areexpressed, and the use of said antibodies for characterizing the precisecellular localization of said C. pneumoniae proteins. Also within thescope of the present invention is the use of monospecific antibodiesagainst proteins of the invention for determining which part of saidproteins is surface exposed and how proteins in the C. pneumoniae COMCinteract with each other.

Preferred embodiments of the present invention relate to polypeptideswhich comprise subsequences of the proteins of the invention, saidsubsequences comprising the sequence GGAI. Further preferred embodimentsof the present invention relate to polypeptides which comprisesubsequences of the proteins of the invention, said subsequencescomprising the sequence FSGE.

Polypeptides according to the invention will typically be of a length ofat least 6 amino acids, preferably at least 15 amino acids, preferablyat least 20 amino acids, preferably at least 25 amino acids, preferablyat least 30 amino acids, preferably at least 35 amino acids, preferablyat least 40 amino acids, preferably at least 45 amino acids, preferablyat least 50 amino acids, preferably at least 55 amino acids, preferablyat least 100 amino acids.

A very important aspect of the present invention relates to nucleic acidfragments of the invention derived from Chlamydia pneumoniae, variantsand subsequences thereof.

Another important aspect of the present invention relates to antibodiesagainst the proteins according to the invention, such antibodiesincluding polyclonal monospecific antibodies and monoclonal antibodiesagainst proteins with sequences selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, and SEQ ID NO: 24.

A very important aspect of the present invention relates to diagnostickits for the diagnosis of infection of a mammal, such as a human, withChlamydia pneumoniae, said kits comprising one or more proteins withamino acid sequences selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18; SEQ ID NO: 20, SEQ ID NO:22, and SEQ ID NO: 24.

Another very important aspect of the present invention relates todiagnostic kits for the diagnosis of infection of a mammal, such as ahuman, with Chlamydia pneumoniae, said kits comprising antibodiesagainst a protein with an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24. Antibodies includedin a diagnostic kit according to the invention can be polyclonal ormonoclonal or a mixture hereof.

Still another very important aspect of the present invention relates todiagnostic kits for the diagnosis of infection of a mammal, such as ahuman, with Chlamydia pneumoniae, said kits comprising one or morenucleic acid fragments with sequences selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, and SEQ ID NO: 23.

An aspect of the present invention relates to a composition forimmunizing a mammal, such as a human, against Chlamydia pneumoniae, saidcomposition comprising one or more proteins with amino acid sequencesselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24.

An important role for the proteins of the invention in prevention ofinfection of a mammal, such as a human, with C. pneumoniae is expected.Thus proteins of the invention, including variants and subsequences willbe produced, typically by using recombinant techniques, and will then beused as an antigen in immunization of mammals, such as rabbits.Subsequently, the hyper immune sera obtained by the immunization will beanalyzed for protection against C. pneumoniae infection using a tissueculture assay. In addition it is contemplated that monoclonal antibodieswill be produced, typically using standard hybridoma techniques, andanalyzed for protection against infection with C. pneumoniae.

It is envisioned that particularly interesting and immunogenic epitopeswill be found in connection with the proteins of the invention, whichwill comprise subsequences of said proteins. It is preferred to usepolypeptides comprising such subsequences of the proteins of theinvention in immunizing a mammal, such as a human, against Chlamydiapneumoniae.

An important aspect of the present invention relates to the use ofproteins with sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, and SEQ ID NO: 24 in diagnosis of infection of a mammal, such asa human, with Chlamydia pneumoniae.

A preferred embodiment of the present invention relates to the use ofproteins according to the invention in an undenatured form, in diagnosisof infection of a mammal, such as a human, with Chlamydia pneumoniae.

A very important aspect of the present invention relates to the use ofproteins with sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ IDNO: 22, and SEQ ID NO: 24, for immunizing a mammal, such as a human,against Chlamydia pneumoniae.

A preferred embodiment of the present invention relates to the use ofproteins according to the invention in an undenatured form, forimmunizing a mammal, such as a human, against Chlamydia pneumoniae.

A very important aspect of the present invention relates to the use ofnucleic acid fragments with nucleotide sequences selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23 for immunizing amammal, such as a human, against Chlamydia pneumoniae.

It is envisioned that one type of vaccine against C. pneumoniae will bedeveloped by using gene-gun vaccination of mice. Typically, differentgenetic constructs containing nucleic acid fragments, combinations ofnucleic acid fragments according to the invention will be used in thegene-gun approach. The mice will then subsequently be analyzed forproduction of both humoral and cellular immune response and forprotection against infection with C. pneumoniae after challengeherewith.

In line with this, the invention also relates to the uses of theproteins of the invention as a pharmaceutical (a vaccine) as well as tothe uses thereof for the preparation of a vaccine against infectionswith Chlamydia pneumoniae.

Preparation of vaccines which contain protein sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior, to injection may also be prepared. The preparation mayalso be emulsified. The active immunogenic ingredient is often mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredient. Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol, or the like, and combinationsthereof. In addition, if desired, the vaccine may contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, or adjuvants which enhance the effectiveness of thevaccines.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10-95%of active ingredient, preferably 25-70%, and optionally a suitablecarrier.

The protein sequences may be formulated into the vaccine as neutral orsalt forms known in the art. The vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective and immunogenic. The quantity to beadministered depends on the subject to be treated. Suitable dosageranges are of the order of several hundred micrograms active ingredientper vaccination with a preferred range from about 0.1 μg to 1000 μg. Theimmune response may be enhanced if the vaccine further comprises anadjuvant substance as known in the art. Other possibilities involve theuse of immunomodulating substances such as lymphokines (e.g. IFN-γ, IL-2and IL-12) or synthetic IFN-γ inducers such as poly I:C in combinationwith the above-mentioned adjuvants.

It is also possible to produce a living vaccine by introducing, into anon-pathogenic microorganism, at least one nucleic acid fragmentencoding a protein fragment or protein of the invention, and effectingexpression of the protein fragment or the protein on the surface of themicroorganism (e.g. in the form of a fusion protein including a membraneanchoring part or in the form of a slightly modified protein or proteinfragment carrying a lipidation signal which allows anchoring in themembrane). The skilled person will know how to adapt relevant expressionsystems for this purpose.

Another part of the invention is based on the fact that recent researchhave revealed that a DNA fragment cloned in a vector which isnon-replicative in eukaryotic cells may be introduced into an animal(including a human being) by e.g. intramuscular injection orpercutaneous administration (the so-called “gene gun” approach). The DNAis taken up by e.g. muscle cells and the gene of interest is expressedby a promoter which is functioning in eukaryotes, e.g. a viral promoter,and the gene product thereafter stimulates the immune system. Thesenewly discovered methods are reviewed in Ulmer et al., 1993, whichhereby is included by reference.

Thus, a nucleic acid fragment encoding a protein or protein of theinvention may be used for effecting in vivo expression of antigens, i.e.the nucleic acid fragments may be used in so-called DNA vaccines. Hence,the invention also relates to a vaccine comprising a nucleic acidfragment encoding a protein fragment or a protein of the invention, thevaccine effecting in vivo expression of antigen by an mammal, such as ahuman, to whom the vaccine has been administered, the amount ofexpressed antigen being effective to confer substantially increasedresistance to infections with Chlamydia pneumoniae in an mammal, such asa human.

The efficacy of such a “DNA vaccine” can possibly be enhanced byadministering the gene encoding the expression product together with aDNA fragment encoding a protein which has the capability of modulatingan immune response. For instance, a gene encoding lymphokine precursorsor lymphokines (e.g. IFN-γ, IL-2, or IL-12) could be administeredtogether with the gene encoding the immunogenic protein fragment orprotein, either by administering two separate DNA fragments oradministering both DNA fragments included in the same vector. It is alsoa possibility to administer DNA fragments comprising a multitude ofnucleotide sequences which each encode relevant epitopes of the proteinfragments and proteins disclosed herein so as to effect a continuoussensitization of the immune system with a broad spectrum of theseepitopes.

The following experimental non-limiting examples are intended toillustrate certain features and embodiments of the invention.

LEGENDS TO FIGURES

FIG. 1. The figure shows electron microscopy of negative stainedpurified C. pneumoniae EB (A) and purified OMC (B).

FIG. 2. The figure shows silver stained 15% SDS-PAGE of purified EB andOMC. Lane 1, purified C. pneumoniae EB; lane 2, C. pneumoniae OMC; lane3, purified C. trachomatis EB; and lane 4 C. trachomatis OMC.

FIG. 3. The figure shows immunoblotting of C. pneumoniae EB separated by10% SDS-PAGE, transferred to nitrocellulose and reacted with rabbit antiC. pneumoniae OMC.

FIG. 4. The figure shows coomassie blue stained 7.5% SDS-PAGE ofrecombinant pEX that were detected by the rabbit anti C. pneumoniaeserum. Arrow indicated the localization of the 117 kDa b-galactosidaseprotein.

FIG. 5. The figure shows immunoblotting of recombinant pEX colonesdetected by colony blotting separated by 7.5% SDS-PAGE and transferredto nitrocellulose and reacted with rabbit anti C. pneumoniae OMC. Lane1, seablue molecular weight standard. Lane 2-6 pEX clones cultivated at42° C. to induce the production of the b-galactosidase fusion proteins.

FIG. 6. The figure shows sequence strategy for Omp4 and Omp5. Arrowsindicates primers used for sequencing.

FIG. 7. C. pneumoniae omp genes. The genes are arranged in two clusters.In cluster 1 Omp12, 11, 10, 5, 4, 13, and 14 are found. In cluster 2 arefound Omp6, 7, 8, 9, and 15.

FIG. 8 A-J. The figure shows alignment of C. pneumoniae Omp4-15, usingthe program pileup in the GCG package.

FIG. 9. The figure shows immunofluorescence of C. pneumoniae infectedHeLa, 72 hrs. after infection, reacted with mouse monospecificanti-serum against pEX3-36 fusion protein. pEX3-36 is a part of the Omp5gene.

FIG. 10. The figure shows immunoblotting of C. pneumoniae EB, lane 1-3heated to 100° C. in SDS-sample buffer, lane 4-6 unheated. Lane 1reacted with rabbit anti C. pneumoniae OMC; lane 2 and 4 pre-serum; lane3 and 5 polyclonal rabbit anti pEX1-1 fusion protein; lane 6 MAb 26.1.

FIG. 11. The figure shows immunoblotting of C. pneumoniae EB, lane 1-4heated to 100° C. in SDS-sample buffer, lane 5-6 unheated. Reacted withserum from C57-black-mice 14 days after infection with 10⁷ CFU of C.pneumoniae. Lane 1 and 5 mouse 1; lane 2 and 6 mouse-2; lane 3 and 5mouse 3; and lane 4H and 8 mouse 4.

FIG. 12. The figure shows immunohistochemistry analysis of mouse lungtissue with C. pneumoniae inclusions present both in the bronchialepithelium and in the lung parenchyma (arrows).

EXAMPLE 1 Cloning of the Genes Encoding the 98/95 kDa C. pneumoniae COMCProteins

Purification of C. pneumonia EBs and COMC

C. pneumoniae was cultivated in HeLa cells. Cultivation was doneaccording to the specifications of Miyashita and Matsumoto (1992), withthe modification that centrifugation of supernatant and of the laterprecipitate and turbid bottom layer was carried out at 100,000×g. Themicroorganism attached to the HeLa cells by 30 minutes of centrifugationat 1000×g, after which the cells were incubated in RPMI 1640 medium(Gibco BRL, Germany cat No. 51800-27), containing 51 foetal calf serum(FCS, Gibco BRL, Germany Cat No. 10106.169) gentamicin for two hours at37° C. in 5% CO2 atmosphere. The medium was changed to medium that inaddition contained 1 mg per ml of cycloheximide. After 48 hours ofincubation a coverslip was removed from the cultures and the inclusionwas tested with an antibody specific for C. pneumoniae (MAb 26.1)(Christiansen et al. 1994) and a monoclonal antibody specific for thespecies C. trachomatis (MAb 32.3, Loke diagnostics, Århus Denmark) toensure that no contamination with C. trachomatis had occurred. The HeLacells were tested by Hoechst stain for Mycoplasma contamination as wellas by culture in BEa and BEg medium (Freund et al., 1979). Also the C.pneumoniae stocks were also tested for Mycoplasma contamination bycultivation in BEa and BEg medium. No contamination with C. trachomatis,Mycoplasmas or bacteria were detected in cultures or cells. 72 hourspost-infection the monolayer was washed in PBS, the cells were loosenedin PBS with a rubber policeman, and the Chlamydia were liberated fromthe host cell by sonication. The C. pneumoniae EBs and RBs were purifiedon discontinuous density gradients (Miyashita et al. (1992)). The purityof the Chlamydia EBS were verified by negative staining andelectronmicroscopy (FIG. 1), only particles of a size of 0.3 to 0.5 mmwere detected in agreement with the structure of C. pneumonia EBs. Thepurified Chlamydia EBs were subjected to sarkosyl extraction asdescribed by Caldwell et al (1981) with the modification that a briefsonication was used to suspend the COMC. The purified COMC was tested byelectronmicroscopy and negative staining (FIG. 1), where a folded outermembrane complex was seen.

SDS-PAGE Analysis of Purified EBs and COMC

The proteins from purified EBs and C. pneumoniae OMC were separated on15% SDS-polyacrylamide gel, and the gel was silver stained (FIG. 2), inlane 1 it is seen that the purified EBs contain major proteins of 100/95kDa and a protein of 38 kDa, in the purified COMC (lane 2) these twoprotein groups are also dominant. In addition, proteins with a molecularweight of 62/60 kDa, 55 kDa, and 12 kDa have been enriched in the COMCpreparation. When the purified C. pneumoniae EB is are compared topurified C. trachomatis EB (lane 3) it is seen that predominant proteinin the C. trachomatis EB is the major outer membrane protein (MOMP), andit is also the dominant band in the COMC preparation of C. trachomatis(lane 4), and Omp2 of 60/62 kDa as well as Omp3 at 12 kDa are seen inthe preparation. However, no major bands with a size of 100/95 kDa aredetected as in the C. pneumoniae COMC preparation.

Production of Rabbit Polyclonal Antibodies Against C. pneumoniae COMC

To ensure production of rabbit antibodies that would recognize all theC. pneumoniae proteins in immuno-blotting and colony-blotting 10 μg ofCOMC antigen was dissolved in 20 μl of SDS sample buffer and thereafterdivided into 5 vials. The dissolved antigen was further diluted in oneml of PBS and one ml of Freund incomplete adjuvant (Difco laboratories,USA cat. No. 0639-60-6) and injected into the quadriceps muscle of a NewZealand white rabbit. The rabbit was given three times intramuscularinjections at an interval of one week, and after further three weeks thedissolved COMC protein, diluted in one ml PBS was injectedintravenously, and the procedure was repeated two weeks later. Elevenweeks after the beginning of the immunization, the serum was obtainedfrom the rabbit. Purified C. pneumoniae EBs were separated by SDS-PAGE,and the proteins were electrotransferred to nitrocellulose membrane. Themembrane was blocked and immunostained with the polyclonal COMC antibody(FIG. 3). The serum recognized proteins with a size of 100/95, 60 and 38kDa in the EB preparation. This is in agreement with the sizes of theouter membrane proteins.

Cloning of the COMC Proteins

Due to the cultivation of C. pneumoniae in HeLa cells, contaminatinghost cell DNA could be present in the EB preparations. Therefore, thepurified EB preparations were treated with DNAse to remove contaminatingDNA. The C. pneumoniae DNA was then purified by CsCl gradientcentrifugation. The C. pneumoniae DNA was partially digested with Sau3Aand the fractions containing DNA fragments with a size of approx. 0.5 to4.0 kb were cloned into the expression vector system pEX (Boehringer,Germany cat. No. 1034 766, 1034 774, 1034 782). The pEX vector systemhas a β-galactosidase gene with multiple cloning sites in the 3′ end ofthe β-galactosidase gene. Expression of the gene is regulated by the PRpromoter, so the protein expression can be induced by elevating thetemperature from 32 to 42° C. The colonies of recombinant bacteria weretransferred to nitrocellulose membranes, and the temperature wasincreased to 42° C. for two hours. The bacteria were lysed by placingthe nitrocellulose membranes on filters soaked in 5% SDS. The coloniesexpressing outer membrane proteins were detected with the polyclonalantibody raised against C. pneumoniae COMC. The positive clones werecultivated in suspension and induced at 42° C. for two hours. Theprotein profile of the clones were analysed by SDS-PAGE, and increasesin the size of the induced b-galactosidase were observed (FIG. 4). Inaddition, the proteins were electrotransferred to nitrocellulosemembranes, and the reaction with the polyclonal serum against COMC wasconfirmed (FIG. 5).

Sequencing of Positive COMC Clones

To characterize the pEX clones, the inserted C. pneumoniae DNA wassequenced. The resulting DNA sequences were searched against theprokaryotic sequences in the GenEmbl database. The search identified 6clones as part of the Omp2 gene, and 2 clones as part of the Omp3 gene,and 2-clones as part of the MOMP gene, indicating that COMC proteins hadbeen successfully cloned. Furthermore, 32 clones were obtained,containing DNA sequences not found in the GenEmbl database. Thesesequences could, however, be clustered in two contics of 6 and 4 clones,and three clones were identical. In addition 19 clones were found withno overlap to the contics (FIG. 7). To obtain more sequence data for thegenes, C. pneumoniae DNA was totally digested with BamHI restrictionenzyme, and the fragments were cloned into the vector pBluescript. Theligated DNA was electrotransformed into E. coli XL1-Blue and selected onplates containing Ampicillin. The recombinant bacterial colonies weretransferred to a nitrocellulose membrane, and colony hybridisation wasperformed using the inserts of pEX 1-1 clone as a probe. A clonecontaining a single BamHI fragment of 4.5 kb was found, and thehybridisation to the probe was confirmed by Southern blotting. Theinsert of the clone was sequenced bi-directionally using syntheticprimers for approx. each 300 bp. The sequence of the BamHI fragment madeit possible to join the two contics of pEX clones. Totally, togetherwith the pEX clones it was possible to assemble 6.5 kb DNA sequence,encoding two new COMC proteins. (FIG. 6)

Additional sequences were obtained by PCR performed on purified C.pneumoniae DNA with primers both from the known Omp genes and from otherknown genes. The obtained PCR products were sequenced, The sequenceorganisation is shown in FIG. 7. Additional 8 Omp genes were detected.The alignment of the deduced amino acid sequences are shown in FIGS. 8 Aand B.

Analysis of DNA Sequence

The DNA sequence encoding the Omp4-15 proteins with a size of 89.6-100.3kDa (and for Omp13: 56.1 kDa). Omp4 and Omp5 were transcribed inopposite directions. Downstream Omp4 a possible termination structurewas located. The 3′ end of the Omp5 gene was not cloned due to thepresence of the BamHI restriction enzyme site positioned within thegene. The translated DNA sequence of Omp4 and Omp5 was compared by useof the gap programme in the GCG package (Wisconsin package, version8.1-UNIX, August 1995, sequence analysis software package). The twogenes had an amino acid identity of 41% (similarity 61%), and a possiblecleavage site for signal peptidase 1 was present at amino acid 17 inOmp4 and amino acid 25 in Omp5. When the amino acid sequence encoded bytwo other pEX clones were compared to the sequence of Omp4 and Omp5 theyalso, had amino acid homology to the genes. It is seen that the twoclones have homology to the same area in the Omp4 and Omp5 proteins.Consequently, the pEX clones must have originated from two additionalgenes. Therefore these genes were named Omp6 and Omp7. Similar analyseswere performed with the other genes. In contrast to what was seen forOmp4 and 5 none of the other putative omp proteins had a cleavage sitefor signal peptides.

EXAMPLE 2 Polyclonal Monospecific Antibodies Against pEX Fusion Proteinsand Full Length Recombination+Omp4

To investigate the topology of the Omp4-7 proteins, representative pEXclones, were selected from each gene. The fusion proteins ofβ-galactosidase/omp were induced, and the proteins were partiallypurified as inclusion bodies. Balb/c mice were immunized three timesintramuscular with the antigens at an interval of one week, and, aftersix weeks the serum was obtained from the mice. HeLa cells were infectedwith the C. pneumoniae. 72 hours after the infection the mono-layerswere fixed with 3.7% formaldehyde. This treatment makes the outermembrane of the Chlamydia impermeable for antibodies due to theextensive cross-linking of the outer membrane proteins by theformaldehyde. The HeLa cells were permeabilized with 0.2% Triton X100,the monolayers were washed in PBS, then incubated with 20% (v/v) FCS toinactivate free radicals of the formaldehyde. The mice sera were diluted1:100 PBS with 20% (v/v) FCS and incubated with the monolayers for halfan hour. The monolayers were washed in PBS and secondary FITCHconjugated rabbit antimouse serum was added for half an hour, and themonolayers were washed and mounted. Several of the antibodies reactedstrongly with the EBs in the inclusions (FIG. 9). In spite of theformaldehyde fixation it could not be excluded that the surface of theEB was changed by the treatments, so that the antibodies could getaccess to the Omp4-7. Therefore, the reaction was confirmed byimmuno-electron microscopy with the antibody raised against clonepEX3-36. Purified EB of C. pneumoniae were absorbed to carbon coatednickel-grids. After the absorption the grids were washed with PBS andblocked in 0.5% Ovalbumin dissolved it PBS. The antibodies were diluted1:100 in the same buffer and incubated for 30 minutes. The grids werewashed in PBS. Rabbit anti mouse Ig conjugated with 10 nm colloidal golddiluted in PBS containing 1% gelatin was added to the grids for half anhour. The grids were washed in 3×PBS with 1% gelatin and 3 times in PBS,the grids were contrastained with 0.7% phospho tungstic acid. The gridswere analysed in a Jeol 1010 electron microscope at 40 kV. It was seenthat the gold particles were covering the surface of the purified EB.Because the C. pneumoniae EBs were not exposed to any detergent orfixation under either the purification or the reaction with antibodies,these results show that the cloned proteins have surface exposedepitopes.

Polyclonal Monospecific Antibodies Against Omp4

The Omp4 gene was amplified by PCR with primers that containedLIC-sites, and the PCR product was cloned into the pET-30 LIC vector(Novagen). The histidine tagged fusion protein was expressed byinduction of the synthesis by IPTG and purified over a nickel column.The purified Omp4 protein was used for immunization of a rabbit (sixtimes, 8 μg each time).

Use of Rabbit Polyclonal Antibodies to Recombinant Omp4 for Detection ofChlamydia pneumoniae in Paraffin Embedded Sections

The lungs of C. pneumoniae infected mice were obtained three days afterintranasal infection. The tissue samples were fixed in 4% formaldehyde,paraffin embedded, sectioned and deparaffinized prior to staining. Thesections were incubated with the rabbit serum diluted 1:200 in TBS (150μM NaCl, 20 mM Tris pH 7.5) for 30 min at room, temperature. After washtwo times in TBS the sections were incubated with the secondary antibody(biotinylated goat anti-rabbit antibodies) diluted 1:300 in TBS,followed by two times wash in TBS. The sections were stained withstreptavidin-biotin complex (streptABComplex/AP, Dako) for 30 min washedand developed under microscopic inspection with chromagen+new fuchsin(Vector laboratories). The sections were counter stained withHematoxylin and analyzed ny microscopy.

Immuno Blotting Analysis with Hyperimmune Monospecific Rabbit Anti-Serum

The insert of pEX1-1 clone was amplified by PCR using primers containingLIC sites. The PcR product could therefore be inserted in the pET-32 LICvector (Novagen, UK cat No. 69076-1). Thereby the insert sequence of thepEX1-1 clone was expressed in the new vector as a fusion protein, thepart of the fusion protein encoded by the pET-32 LIC vector had 6histidine residues in a row. The expression of the fusion protein wasinduced in this vector, and the fusion protein could be purified underdenaturing condition on a Ni2+ column due to the high affinity of thehistidine residues to divalent cations. The purified protein was usedfor immunization of a New Zealand white rabbit. After 6 timesintramuscular and 2 times intravenous immunization the serum wasobtained from the rabbit. Purified C. pneumoniae EB was dissolved inSDS-sample buffer. Half of the sample was heated to 100° C. in thesample buffer, whereas the other half of the sample was not heated. Thesamples were separated by SDS-PAGE, and the proteins were transferred tonitrocellulose, the serum was reacted with the strips. With the samplesheated to 100° C. the serum recognized a high molecular weight band ofapproximately 98 kDa. This is in agreement with the predicted size ofOmp5, of which the pEX1-1 clone is a part, however; when the antibodywas reacted to the strip, with unheated EB, the pattern was different.Now a band was seen with a size of 75 kDa, in addition weaker bands wereobserved above the band (FIG. 10). These data demonstrate that Omp5needs boiling in SDS-sample buffer to be fully denatured and migratewith a size as predicted from the gene product. When the samples werenot boiled, the protein was not fully denatured and less SDS binds tothe protein and it has a more globular structure that will migratefaster in the acrylamide gel. The band pattern looked identical to whatwas obtained with a monoclonal antibody (MAb 26.1) (lane 6), we earlierhave described (Christiansen et al., 1994), reacting with the surface ofC. pneumoniae EB, but the antibody do not react with the fully SDSdenatured C. pneumoniae. EB in immunoblotting.

Experimental Infection of C57 Black Mice

Due to the realization of the altered migration of the Omp4-7 proteinswithout boiling, we chose to analyse antibodies against C. pneumoniaeEBs after an experimental infection of mice. To obtain antibodies froman infection caused by C. pneumoniae, C57 black mice were inoculatedintranasally with 10⁷ CFI of C. pneumoniae under a light etheranaesthesia. After 14 days of infection the serum samples were obtainedand the lungs were analysed for pathological changes. In two of the micea severe pneumonia was observed in the lung sections, and in the thirdmouse only minor changes were observed. The serum from the mice wasdiluted 1:100 and reacted with purified EBs dissolved in sample bufferwith and without boiling. In the preparations that had been heated to100° C. the sera from two of the mice reacted strongly with bands of60/62 kDa and weaker bands of 55 kDa, but no reaction was observed withproteins of the size of Omp4-7 (FIG. 11). However, when the sera werereacted with the preparation that had not been heated they all had astrong reaction with a-broad band of an approximate size of 75 kDa. Thisis in agreement with the size of the Omp4-7 proteins in the unheatedpreparation. Therefore, it could be concluded that the epitopes of theOmp4-7 proteins recognized by the antibodies after a C. pneumoniaeinfection were discontinuous epitopes because the full denaturation ofthe antigen completely destroyed the epitopes. The 75 kDa proteinobserved in unheated samples is not Omp2 (Shown in immunoblotting withan Omp2 specific antibody)

EXAMPLE 3 Comparison of Omp4-7 of C. pneumoniae with Putative OuterMembrane Proteins (POMP) of C. psittaci

Longbottom et al. (1996) have published partial sequence from 98 to 90kDa proteins from C. psittaci. They have entered the full sequence of 5genes in this family in the EMBL database. They have named the genes“putative outer membrane proteins” (POMP) since their precise locationwas not determined. The family is composed of two genes that arecompletely identical, and two genes with high homology to these genes.They calculated a molecular size of 90 and 91 kDa. The 5th encode aprotein of 98 kDa. The sequence of the Omp4-7 proteins of C. pneumoniaewere compared to the sequences of the C. Psittaci POMP proteins with theprogramme pileup in the GCG package. The amino acid homologies were inthe range of 51-63%. It is seen that the C. pneumoniae-Omp4-5 proteinsare most related to the 98 kDa POMP protein of C. psittaci.Interestingly, the 98 kDa C. psittaci POMP-protein is more related tothe C. pneumoniae genes than to the other C. psittaci-genes. Therepeated sequences of GGAI were conserved in the 98 kDa POMP protein,but only three GGAI repeats were present in the 90 and 91 kDa C.psittaci POMP proteins. For C. psittaci it has been shown thatantibodies to these proteins seem to be protective for the infection.

REFERENCES

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1. A species specific method for identifying infection of a mammal withChlamydia pneumoniae, said method comprising detecting in a patient orin a patient sample the presence of antibodies against one or moreproteins from the outer membrane of Chlamydia pneumoniae, said proteinsbeing outer membrane proteins having the sequences as shown in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, orSEQ ID NO:24, or a variant or subsequence thereof which is recognized bya species-specific antibody which specifically binds said outer membraneprotein.
 2. Method according to claim 1 wherein detection of nucleicacid fragments is obtained by using nucleic acid amplification. 3.Method according to claim 2, wherein detection of nucleic acid fragmentsis obtained by using polymerase chain reaction.
 4. A nucleic acidfragment derived from Chlamydia pneumoniae comprising the nucleotidesequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, or SEQ ID NO:23, or a variant or subsequence ofsaid nucleotide sequence which has a sequence homology of at least 50%with any of the sequences mentioned and wherein a subsequence of 100nucleic acids or lower shows a homology of at least 80%.
 5. Polyclonalmonospecific antibody against the protein with the sequence shown in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, or SEQ ID NO:24, or a variant or subsequence thereof.
 6. Adiagnostic kit for the diagnosis of infection of a mammal, such as ahuman, with Chlamydia pneumoniae, said kit comprising antibodiesaccording to claim
 5. 7. A diagnostic kit for the diagnosis of infectionof a mammal with Chlamydia pneumoniae, said kit comprising a nucleicacid fragment according to claim
 4. 8. A method of immunizing a mammalagainst Chlamydia pneumoniae which comprises use of an immunologicallyeffective amount of an outer membrane protein having the sequence shownin SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, or SEQ ID NO:24, or a variant or subsequence thereof which isrecognized by a species-specific antibody which specifically binds saidouter membrane protein, for immunizing a mammal against Chlamydiapneumoniae.
 9. The method of claim 6 wherein the protein is inundenatured form.
 10. A method of immunizing a mammal against Chlamydiapneumoniae which comprises use of an immunologically effective amount ofa nucleic acid fragment according to claim 4, encoding a protein whichcomprises one or more protective epitopes of a Chlamydia pneumoniaeantigen, to immunize a mammal, by administering said nucleic acidfragment under conditions conducive to expression of said protein andsubsequent immunization of said mammal by said protein.